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Plant anther specific promoter PCHF15 and application thereof

An anther-specific and promoter technology, applied in the fields of genetic engineering and molecular biology, to achieve precise expression levels

Active Publication Date: 2017-02-22
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few pollen and anther-specific promoters identified in plants, and further identification and discovery of pollen-specific promoters are needed

Method used

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  • Plant anther specific promoter PCHF15 and application thereof
  • Plant anther specific promoter PCHF15 and application thereof
  • Plant anther specific promoter PCHF15 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Acquisition of rice anther-specific promoter PCHF15

[0042] 1. Extraction of rice genomic DNA

[0043] Rice genomic DNA was extracted according to the method instructions of the Plant DNA Isolation Kit (Chengdu Fuji Biotechnology Co., Ltd.). The genome was derived from fresh leaves of rice Nipponbare variety. The extracted genomic DNA was aliquoted and stored at -20°C for later use.

[0044] 2. Design PCR PCHF15 primers

[0045] Primers were designed using the Gibson Assembly method, and the amplified product was inserted into the NcoI and HindIII restriction sites of the 1300GUSplus vector. Primers for amplifying the PCHF15 gene are shown in the sequences SEQ ID NO.2 and SEQ ID NO.3. Among them, about 15 nucleotide sequences at the 5' ends of the upstream and downstream primers were repeated with the corresponding connection positions of the vector to facilitate Gibson Assembly connection.

[0046] PCR reaction system (100μL):

[0047] DNA template: 3...

Embodiment 2

[0058] Example 2: Construction of the recombinant expression vector 1300gus-PCHF15 of the promoter PCHF15

[0059] See the build process figure 1 , Insert the amplified product of Example 1 into the 1300GUSplus vector NcoI and HindIII restriction sites.

[0060] Primers SEQ ID NO.2 and SEQ ID NO.3 amplify the PCR product and recover a product of about 2000 bp by 1% agarose gel electrophoresis. The vector 1300GUSplus was digested with NcoI and HindIII, and the linearized vector was recovered.

[0061] 2X ligation kit ligates promoter PCHF15 to 1300GUSplus, 10μL system is as follows:

[0062] 2.5 μL PCHF15PCR product (50ng)

[0063] 2.5μL enzyme-cut carrier (100ng)

[0064] 5μL Ligation Mix

[0065] Ligation procedure: 50°C 60min.

[0066] Transformation: Take 5 μL of the ligation product above to transform Escherichia coli competent cells by electroporation. Select positive clones for sequencing, the results are as follows image 3 shown. Named 1300gus-PCHF15.

[0067] ...

Embodiment 3

[0068] Example 3: Construction of recombinant expression vector DX2182-PCHF15 containing PCHF15 promoter sequence

[0069] refer to figure 2 The construction process of this method is to construct the recombinant cloning vector pGEM-PCHF15 containing the PCHF15 promoter sequence first, and then construct the recombinant expression vector DX2182-PCHF15 containing the PCHF15 promoter sequence based on it.

[0070] 1. Construction of recombinant cloning vector pGEM-PCHF15 containing PCHF15 promoter sequence

[0071] With reference to the method of Example 1, the primer pair shown in sequence SEQ ID NO.2 and SEQ ID NO.3 is amplified with sequence such as the primer pair shown in SEQ ID NO.5 and SEQ ID NO.6. The obtained amplification product was connected to the pGEM-T vector, and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector pGEM-PCHF15. Transform Escherichia coli com...

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Abstract

The invention relates to genetic engineering and molecular biology, and particularly discloses a plant anther specific promoter PCHF15 and application thereof. The invention further provides an expression vector with the plant anther specific promoter PCHF15, a genetic expression box and a carrier with the genetic expression box. The invention also provides a primer pair for amplifying the plant anther specific promoter PCHF15. The plant anther specific promoter PCHF15 is an endogenous gene of rice, is beneficial to genetic engineering of the rice, and drives exogenous genes to accurately express specificity in pollen, so that a novel method for driving specificity expression of the exogenous genes in the pollen is provided.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a plant anther-specific promoter PCHF15 and its application. Background technique [0002] Plant genes are mainly regulated at the transcriptional level, which is coordinated between various cis-acting elements and trans-acting factors. The promoter is an important regulatory element in plant gene transcription, generally located in the upstream region of the 5' end of the structural gene, and is the recognition and binding site of RNA polymerase and some trans-acting factors. Promoter is the regulatory center of gene transcription. In-depth study of promoter is not only conducive to mastering the control of gene transcription mode and regulatory mechanism, but also conducive to the regulation of exogenous gene expression. The promoter mainly has two regions, which are the core promoter region and the transcriptional regulatory region. The core promoter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/82A01H5/02
CPCC12N15/113C12N15/8231
Inventor 黄培劲张维吴春瑜陈思兰吴永忠
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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